Characterization of a thermostable DNA glycosylase specific for U/G and T/G mismatches from the hyperthermophilic archaeon Pyrobaculum aerophilum

J Bacteriol. 2000 Mar;182(5):1272-9. doi: 10.1128/JB.182.5.1272-1279.2000.

Abstract

U/G and T/G mismatches commonly occur due to spontaneous deamination of cytosine and 5-methylcytosine in double-stranded DNA. This mutagenic effect is particularly strong for extreme thermophiles, since the spontaneous deamination reaction is much enhanced at high temperature. Previously, a U/G and T/G mismatch-specific glycosylase (Mth-MIG) was found on a cryptic plasmid of the archaeon Methanobacterium thermoautotrophicum, a thermophile with an optimal growth temperature of 65 degrees C. We report characterization of a putative DNA glycosylase from the hyperthermophilic archaeon Pyrobaculum aerophilum, whose optimal growth temperature is 100 degrees C. The open reading frame was first identified through a genome sequencing project in our laboratory. The predicted product of 230 amino acids shares significant sequence homology to [4Fe-4S]-containing Nth/MutY DNA glycosylases. The histidine-tagged recombinant protein was expressed in Escherichia coli and purified. It is thermostable and displays DNA glycosylase activities specific to U/G and T/G mismatches with an uncoupled AP lyase activity. It also processes U/7,8-dihydro-oxoguanine and T/7,8-dihydro-oxoguanine mismatches. We designate it Pa-MIG. Using sequence comparisons among complete bacterial and archaeal genomes, we have uncovered a putative MIG protein from another hyperthermophilic archaeon, Aeropyrum pernix. The unique conserved amino acid motifs of MIG proteins are proposed to distinguish MIG proteins from the closely related Nth/MutY DNA glycosylases.

Publication types

  • Comparative Study
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Archaeal Proteins / genetics
  • Archaeal Proteins / metabolism*
  • Base Pair Mismatch
  • Carbon-Oxygen Lyases / metabolism
  • DNA Glycosylases*
  • DNA Repair
  • DNA-(Apurinic or Apyrimidinic Site) Lyase
  • Deoxyribonuclease IV (Phage T4-Induced)
  • Enzyme Stability
  • Escherichia coli / genetics
  • Escherichia coli Proteins*
  • Guanine / analogs & derivatives
  • Guanine / metabolism
  • Molecular Sequence Data
  • N-Glycosyl Hydrolases / genetics
  • N-Glycosyl Hydrolases / metabolism*
  • Phylogeny
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / isolation & purification
  • Recombinant Fusion Proteins / metabolism
  • Sequence Alignment
  • Sequence Homology, Amino Acid
  • Temperature
  • Thermoproteaceae / enzymology*
  • Thermoproteaceae / genetics
  • Thymine DNA Glycosylase*
  • Uracil-DNA Glycosidase

Substances

  • Archaeal Proteins
  • Escherichia coli Proteins
  • Recombinant Fusion Proteins
  • 8-hydroxyguanine
  • Guanine
  • Deoxyribonuclease IV (Phage T4-Induced)
  • endonuclease IV, E coli
  • DNA Glycosylases
  • N-Glycosyl Hydrolases
  • Thymine DNA Glycosylase
  • Uracil-DNA Glycosidase
  • mismatch-specific thymine uracil-DNA glycosylase
  • mutY adenine glycosylase
  • Carbon-Oxygen Lyases
  • DNA-(Apurinic or Apyrimidinic Site) Lyase